relaxivity of per gadolinium atom because of an increase in rotational correlation time. On
the other hand, macromolecular MRI contrast agents may show prolonged intravascular
retention due to its bulky molecular volume, it can be used clinically as a blood pool contrast
agent. In addition, when an organ-targeting group, for example, PM is attached to this
macromolecular metal complex, it can be endowed with liver-targeting property [45-58].
Macromolecular liver-targeting Gd(III) chelates have been developed by the
incorporation of Gd-DTPA and pyridoxanine into polyasparamides, dendrimers and
polyester. Relaxivity studies showed that the chelates possessed obviously higher relaxation
effectiveness than that of Gd-DTPA. MR imaging of the liver in rats and experimental data of
biodistribution in mice indicated that they exhibited liver-targeting properties and enhanced
the contrast of MR images in the liver.
Polyester Liver-Targeting MRI Contrast Agents
Water-soluble polyester ligands were synthesized by the polycondensation of
removed by hydrogenation to give polyesters P(DTPA-GLYC), P(DTPA-PETO) and
P(DTPA-DEA). In the same manner, by adding ethylene glycol (EG) monomer into the
chlorocarbonylated and then incorporated into polyesters. The polyester ligands containing
pyridoxamine group thus prepared were further reacted with GdCl3 in water at room
temperature to give the corresponding hepatic-targeting polyester gadolinium complexes
Figure 4. Structural formula of polyester gadolinium complexes.
158 Guo-Ping Yan, Xiao-Yan Wang, Li-Li Mei
Table 1. Molecular weight of polyesters
P(DTPA-GLYC-EG) 8.73 16.8 1.92
P(DTPA-PETO-EG) 9.47 15.9 1.68
Table 2 Experimental data of relaxivity
Gadolinium complexes [Gd3+](mmol· l-1) T1obsd (s) R1(mmol· l-1·s) -1
Gd-DTPA 1.240 0.138±0.0071 5.6
P(Gd-DTPA-GLYC-PM) 1.3048 0.0631±0.0096 11.91
P(Gd-DTPA-PETO-PM) 1.6195 0.0577±0.0052 10.51
P(Gd-DTPA-DEA-PM) 1.5905 0.0531±0.0073 11.65
P(Gd-DTPA-GLYC-EG-PM) 1.8333 0.0441±0.0053 12.20
P(Gd-DTPA-PETO-EG-PM) 1.3643 0.0476±0.0063 15.17
P(Gd-DTPA-DEA-EG-PM) 1.3950 0.0575±0.0076 12.25
Temp: 25 oC; NMR Frequency: 80MHz; T1d =3.23±0.021s.
0 2000 4000 6000 8000 10000 12000 14000 16000 18000
Viability Relative to Control (%)
Figure 5. Cytotoxicity assay of anticancer drugs in L-02 cells.
Relaxivity studies showed that these polyester gadolinium complexes possess higher
relaxation effectiveness than that of the clinically used small molecular gadolinium complex
Gd-DTPA (Table 2). At the concentration (4280μg ml-1) of polyester ligand and its
Vitamin B6 as Liver-targeting Group in Drug Delivery 159
gadolinium complex in the growth medium (RPMI-1640 media (10% foetal bovine serum
(Gibco Co, USA), 100units ml-1 penicilium, 100μg ml-1 streptomycin)) , the viability of
possess low cytotoxicity to L-02 cells (Figure 5).
In comparison to the signal intensity (SI) of the liver in the rat injected of Gd-DTPA(0.1
mmol/kg) and P(Gd-DTPA-PETO) (0.1 mmol/kg) without the liver-targeting group PM, the
signal intensity of the liver in the rat injected of P(Gd-DTPA-PETO-PM) (0.1mmol/kg) was
obviously enhanced, the irradiated portion of the liver was brighter and the demarcation
became clearer at the same time intervals during the detection time. This result illustrated that
P(Gd-DTPA-PETO-PM) can greatly enhance the contrast of MR images of the liver after
injection (Figure 6-8). Thirty minutes after injection of P(Gd-DTPA-PETO-PM), the signal
enhancement of the liver (black cycle) is 176% (Table 3). It is better than that of Gd-DTPA
(119%). On the other hand, P(Gd-DTPA-PETO-PM) has prolonged intravascular duration
time for approximately one hour (127%). These results indicated that polyester gadonilium
complexes containing pyridoxamine group can be targeted to the liver.
A 1 Control B 1 15 min C 1 30min D 1 45 min
D1 are the T1-weighted images of the rat received injection of Gd-DTPA (0.1 mmol/kg, Magnevist)
after 15 min, 30min and 45min.
Table 3. Enhancement (%) in the signal of the liver in different time after injection
Gd-DTPA P(Gd-DTPA-PETO) P(Gd-DTPA-PETO-PM)
160 Guo-Ping Yan, Xiao-Yan Wang, Li-Li Mei
A 2 Control B 2 15 min C 2 30 min
D 2 45 min E 2 60 min F 2 75 min
E2 and F2 are the T1-weighted images of the rat received injection of P(Gd-DTPA-PETO) (0.1
mmol/kg) without the liver-targeting groups PM after 15min, 30min, 45min, 60min and 75min.
A 3 Control B 3 15 min C 3 30 min
D 3 45 min E 3 60 min F 3 75 min
E3 and F3 are the T1-weighted images of the rat received injection of P(Gd-DTPA-PETO-PM) (0.1
mmol/kg) after 15min, 30min, 45min, 60min and 75min.
Vitamin B6 as Liver-targeting Group in Drug Delivery 161
Polyaspartamide Liver-Targeting MRI Contrast Agents
Polyaspartamide is a biologically water-soluble synthetic polymer with a protein-like
structure. It has been used as a plasma extender and a drug carrier and for some other
biomedical applications because it is nontoxic, nonantigenic and degradable in living
The effects of PHEA and PAEA on cell growth and metabolism of HeLa cells in vitro
were determined as a function of polymer concentration and compared to polylysine. The
preliminary results show that over the concentration range tested, the cells incubated with
PHEA and poly-α,β-[N-(2-amino ethy1)-L-aspartamide] (PAEA) retain 55.1% and 61.9%
viabilities, while in the presence of polylysine (PLys), HeLa cells show no viability under the
same concentration (100µg/mL). At a higher concentration (200µg/mL) of PHEA and PAEA,
the cells still retain 62.4% and 49.8% viabilities respectively, relative to control (Figure 9).
Thus polyaspartamides are the good polymeric carriers for MRI contrast agent and drug
controlled release system [11].
0 20 40 60 80 100 120 140 160 180 200
Viability Relative to Control (%)
Figure 9. Cytotoxicity assay of PLys, PHEA and PAEA in HeLa cells.
bis(N-hydroxysuccinimide) ester. The PM-containing DTPA active ester thus obtained was
ligands PHEA-DTPA-PM and PAEA-DTPA-PM. Finally, by the metalation of the ligands
162 Guo-Ping Yan, Xiao-Yan Wang, Li-Li Mei
with gadolinium Gd(Ⅲ), two kinds of polyaspartamide gadolinium complexes were
Relaxivity studies showed that these polyaspartamide gadolinium complexes possess
higher relaxation effectiveness than that of Gd-DTPA. In vitro cytotoxicity assay showed that
polyaspartamide gadolinium complexes have low cytotoxicity. Magnetic resonance imaging
showed that the signal intensity (SI) of the liver in rat injected with PHEA-Gd-DTPA-PM
(the average percent value of linked of polymeric repeat unit in gadolinium complexes
(wt%): Gd 2.65) or PAEA-Gd-DTPA-PM (the average percent value of linked of polymeric
repeat unit in gadolinium complexes (w%): Gd 11.04) was obviously enhanced. Experimental
data of biodistribution in Kunming mice indicated that the rapid decrease of the
polyaspartamide MRI contrast agent in blood, heart and spleen correlated with its increasing
capture by the liver, indicating that these polyaspartamide MRI contrast agents containing
pyridoxamine were taken up specifically by hepatcocytes.
DTPA and pyridoxamine (PM) as a liver-targeting group were both incorporated into
polyaspartamides i. e. (poly-α,β-[N-(2-hydroxyethyl)-L-aspartamide] (PHEA), poly-α,β-[N-
(3-hydroxypropyl)-L-aspartamide] (PHPA), poly-α,β-[N-(2-aminoethy1)-L-aspartamide]
(PAEA) and poly-α,β-[N-(6-aminohexyl)-L- aspartamide] (PAHA) to obtain the
polyaspartamide ligands. The polyaspartamide containing both DTPA ligands and
pyridoxamine groups thus prepared were further reacted with gadolinium chloride to give the
corresponding polyaspartamide gadolinium complexes with different amount of gadolinium
ions PHEA-Gd-DTPA-PM, PHPA-Gd-DTPA-PM, PAEA-Gd-DTPA-PM and PAHA-GdDTPA-PM [12].
The polyaspartamide gadonilium complexes containing pyridoxamine groups possess
obviously higher relaxation effectiveness than that of Gd-DTPA. PHEA-Gd-DTPA-PM (the
average percent value of linked of polymeric repeat unit in gadolinium complexes (mol%):
Gd-DTPA 5.30, PM 0.80) possesses the low intravenous acute toxicity and LD50/7days
(intravenous, mouse) to IRC mice is 5.2g/kg±0.5g/kg.
Table 4. Enhancement (%) in the signal from the liver at different times after injection
Control 100 100 100 100 100 100 100 100 100
2 104 107 126 171 109 113 109 152 150
8 107 126 128 192 117 117 115 137 162
15 114 129 139 173 132 125 130 135 158
30 119 141 141 173 136 133 133 130 150
45 115 135 133 194 159 141 139 128 141
60 114 133 135 198 152 145 144 128 136
75 133 160 192 148 163 137 119 135
90 146 150 195 145 181 130 110 134
105 156 135 213 144 165 122 102 133
120 154 131 184 133 156 117 100 124
Vitamin B6 as Liver-targeting Group in Drug Delivery 163
MR imaging showed that the signal intensities (SI) of the liver in rat injected with low
dosage of PHEA-Gd-DTPA-PM (0.1mmol/kg, 0.075mmol/kg, 0.05mmol/kg and 0.025
mmol/kg) were obviously enhanced in comparison to that of the liver in the rat injected with
Gd-DTPA (0.1mmol/kg) (Figure 10). It greatly enhanced the contrast of MR image of the
liver and provided prolonged intravascular duration in the liver (Table 4). These results
indicated that the polyaspartamide gadonilium complex containing pyridoxamine groups
could be used as the candidate of specific MRI contrast agent for the liver.
are the T1-weighted images of the rats which received injection with PHEA-Gd-DTPA-PM (the average
percent value of linked of polymeric repeat unit in gadolinium complexes (mol%): Gd-DTPA 5.30, PM
0.80, 0.05mmol/kg) after 15min, 60min and 120min. Indicated areas 1 and 2 were used to calculate
contrast enhancements listed in Table 4.
Dendritic Liver-Targeting MRI Contrast Agents
The conjugation of paramagnetic metal chelates to dendrimers is currently being
explored as a new potential macromolecular MRI contrast agents because dendrimers have
some advantages over other polymer carriers including the highly branched structure, low
polydispersity molecule, uniform surface chemistry and high numbers of reactive functional
groups per unit mass and volume for modification [68-80].
A series of liver-targeting dendritic gadolinium complexes were synthesized by
conjugation of diethylenetriaminepentaacetic acid (DTPA) and pyridoxamine to the terminal
amines of the dendrimers with 1,4,7,10-tetraazacyclododecane as the core (Generation: G1.0-
5.0) and chelation with gadoliniumn chloride (Figure 11). These dendrimer-metal chelate
conjugates have high ion relaxivities (of 13.0 - 23.5 (mmol/L)-1·s-1 at 300MHz, 17℃, and pH
of 7.4). MR imaging showed that the signal intensities (SI) of the liver in rats injected with
low doses of G4.0-Gd-DTPA-PM (the average mole ratio of attached Gd-DTPA and PM to
164 Guo-Ping Yan, Xiao-Yan Wang, Li-Li Mei
amine groups on the surface of the dendrimers (mol%): Gd-DTPA 10.84, PM 1.64) were
significantly enhanced (Figure 12). G4.0-Gd-DTPA-PM greatly enhances the contrast of MR
images of the liver, provides prolonged intravascular duration and produces highly contrasted
visualization of blood vessels in the liver. These novel dendritic gadolinium complexes
containing pyridoxamine groups demonstrate liver-targeting properties and show strong
potential as new liver-targeting MRI contrast agents [13].
Figure 11. Structural formula of G2-Gd-DTPA-PM.
C5 30 min D5 60 min E5 120 min
are the T1-weighted images of the rat which received injections of dendritic G4-Gd-DTPA-PM (0.1
mmol/kg) after 15min, 30min, 60min and 120min. Indicated areas I and 2 were used to calculate
contrast enhancements listed in Table 5.
Vitamin B6 as Liver-targeting Group in Drug Delivery 165
Table 5. Enhancement (%) in the signals from the liver at different times after injection
Figure 13. Structural formula of PAMAM-Gd-DTPA-PM.
In addition, diethylenetriaminepentaacetic acid (DTPA) and pyridoxamine (PM) were
also both incorporated to the amine groups on the surface of the ammonia core
poly(amidoamine) dendrimers (PAMAM, Generation 2.0-5.0) to obtain the dendritic ligands
(Figure 13). These dendritic ligands were further reacted with gadolinium chloride to yield
the corresponding dendritic gadolinium complexes. They also have high relaxivity and higher
intravascular retention time, and greatly enhance the contrast of MR images of the liver.
Animal tests showed that small doses of this dendrimer contrast agent could promise a highly
resolved and contrasted visualization of blood vessels. So the results suggest that this new
and powerful class of contrast agents have the potential for diverse and extensive application
in MR imaging for the liver [14].
166 Guo-Ping Yan, Xiao-Yan Wang, Li-Li Mei
LIVER-TARGETING ANTICANCER CONJUGATES
Polymer-based drug delivery systems are used to optimize the therapeutic properties of
drugs and render them safer, more effective and reliable. Moreover, polymeric drugs with
of view. Now the development of biomedical polymers for drug controlled release was laid
emphasis on the temporal control, distribution control and responsive drug delivery systems
One important approach in drug delivery design is that the attached drugs can be targeted
to specific organs, tissues or cells by the incorporation of a drug into a polymer containing
organ or tissue-targeting group or moiety. By this method, the toxic side effects of the drugs
can be suppressed and the distribution of drugs can be improved and reduce the drugs dose
5-Fluorouracil (5-Fu) was chosen as a drug model because its structure and mode of
action are well described and it is widely utilized in cancer chemotherapy. Anticancer
conjugates of 5-fluorouracil and polyaspartamides containing pyridoxamine moiety were
prepared by conjugating anticancer drug 5-fluorouracil and hepatocyte-targeting group
pyridoxamine to the polyaspartamides with different side chains (poly-α,β-[N-(2-
hydroxyethyl)-L-aspartamide] (PHEA), poly-α,β-[N-(2-aminoethy1)-L-aspartamide] (PAEA),
reaction increased, the conversions of 5-Fu in polymeric drugs increased from 5.9wt% to
25.6wt%. Their properties in vitro and in vivo were also evaluated [64].
In vitro drug release properties studies showed that these anticancer conjugates can
sustain in vitro release rate 5-Fu in PBS. A steady release rate of the drug was maintained for
more than 80h (Figure 14). 5-Fu-PHEA-PM and 5-Fu-PHPA-PM were released faster than 5-
Fu-PAEA-PM and 5-Fu-PAHA-PM because the hydrolysis rate of –NCOO- from the side
chains of 5-Fu-PHEA-PM and 5-Fu-PHPA-PM was faster than that of –NCONH- from the
side chains of 5-Fu-PAEA-PM and 5-Fu-PAHA-PM.
In vitro cytotoxicity assay exhibited that polymeric drugs possess low cytotoxicity to the
human liver cells (L-02) than Fluorofur and 5-fluorouracil (Figure 15). FT-207 is a derivative
of 5-fluorouracil and possesses lower toxicity than 5-fluorouracil. At 225µg/ml of anticancer
drugs in the growth medium, the L-02 cells incubated with FT-207 and 5-Fu-PAEA-PM,
respectively, retained above 47.1% and 63.2% viabilities relative to control.
The distribution of PAEA-DTPA in mice tissues at various time points was shown that
the agent was distributed to all the different tissues (such as the blood, liver, lung, heart and
small intestine) showed no tissue-targeting property. It was rapidly cleared by the kidney,
liver and small intestine, with the predominant excretion route of PAEA-DTPA is through
kidney. The distribution of 5-Fu-PAEA-(DTPA)-PM in mice tissues at various time points
was indicated that the majority of the polymeric drug was transferred from the blood into the
liver within 20 min after injection. Very little amount of polymeric drug was found in the
heart, spleen, lung, small intestine, muscle and bone at any time. This result shows the agent
entered the liver from the blood and was excreted by kidneys later. A high content of 5-Fu-
Vitamin B6 as Liver-targeting Group in Drug Delivery 167
PAEA-(DTPA)-PM stayed in the liver for 90 min after injection. The incorporation of
pyridoxamine in the polymers increased the pinocytic uptake by the liver.
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